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Abstract:

The invention generally relates to methods for screening patients for one
or more clinical conditions using a composite assay. According to certain
aspects, methods of the invention involve isolating at least one nucleic
acid from a biological sample obtained from the subject, detecting at
least one sequence mutation and a chromosomal abnormality in the at least
one nucleic acid in a single assay format, and identifying a clinical
condition in said subject when both the sequence mutation and the
chromosomal abnormality are present.

Claims:

1. A method for detection a clinical condition in a subject, said method
comprising the steps of: isolating at least one nucleic acid from a
biological sample obtained from the subject; detecting at least one
sequence mutation and a chromosomal abnormality in the at least one
nucleic acid in a single assay format; and identifying a clinical
condition in said subject when both the sequence mutation and the
chromosomal abnormality are present.

2. The method of claim 1, wherein the at least one nucleic acid is DNA.

3. The method of claim 2, wherein the DNA is genomic DNA.

4. The method of claim 1, wherein the sequence mutation and chromosomal
abnormality occur on the same chromosome.

5. The method of claim 1, wherein the sequence mutation and the
chromosomal abnormality occur on different chromosomes.

6. The method of claim 1, wherein the sequence mutation is a point
mutation.

7. The method of claim 1, wherein the chromosomal abnormality is loss of
heterozygosity.

8. The method of claim 1, wherein the clinical condition is cancer.

9. The method of claim 8, wherein the cancer is bladder cancer.

10. The method of claim 1, wherein the detecting step further comprises
detecting a chemical modification to the nucleic acid.

11. The method of claim 10, wherein the chemical modification to the
nucleic acid comprises hypermethylation.

13. The method of claim 12, wherein the sequencing technique is a single
molecule sequencing technique.

14. The method of claim 1, wherein a sequence mutation in FGFR3 is
detected.

15. The method of claim 1, wherein a chromosomal abnormality in p53 is
detected.

16. The method of claim 15, wherein the chromosomal abnormality is loss
of heterzygosity.

Description:

RELATED APPLICATION

[0001] This application claims the benefit of and priority to provisional
U.S. patent application Ser. No. 61/594,102, filed on Feb. 2, 2012, the
entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention generally relates to methods for screening patients
for one or more clinical conditions using a composite assay.

BACKGROUND

[0003] Clinical screening is desirable to detect a disease or other
clinical condition, such as cancer, inflammation, or autoimmune disease,
prior to the presentation of related symptoms. Such early stage detection
allows for treatment of the disease or condition when treatment is more
effective and less costly. However, early screening may lead to false
positives or false negatives. These incorrect diagnoses can cause undue
stress on the patient in the form of anxiety, physical discomfort or side
effects from a medical treatment that was not needed. Additionally,
incorrect diagnoses may result in the unnecessary use of other medical
resources, or a loss in confidence by the patient for the efficacy of a
needed medical test. In worst case situations, an incorrect diagnosis can
delay necessary treatment for a medical condition and result in a
terminal illness.

[0004] Diagnostic assays based upon the measurement of multiple biomarkers
have been used as a way to increase the accuracy of a diagnostic
screening test. For example, assays have been proposed in which the gene
expression of several genes is measured in order to assess clinical
status. Multiple protein analytes have also been used to screen for the
presence of any of multiple disorders where the diagnosis is unclear.
Oftentimes however, increasing the number of biomarkers measured in a
given screening assay does not provide a significant improvement over the
measurement of a single biomarker.

SUMMARY OF THE INVENTION

[0005] The invention provides methods for assessing clinical condition by
taking into account different types of underlying genetic information as
well as gene expression data. Methods of the invention result in improved
ability to diagnose the presence of a clinical condition or disorder.
Methods of the invention recognize that a single genetic marker type is
insufficient to diagnose and characterize a clinical condition with high
sensitivity and specificity. According to the invention, methods that
comprise multimodal analysis have greater sensitivity and specificity in
the diagnosis and characterization of disease.

[0006] In one embodiment of the invention, the methods of the invention
involve isolating at least one nucleic acid from a biological sample,
using a single assay format to detect a sequence mutation and a
chromosomal abnormality in the at least one nucleic acid, and identifying
a clinical condition in the patient when both a sequence mutation and
chromosomal abnormality are present. The isolated nucleic acid is
preferably DNA (e.g., genomic DNA). The detected sequence mutation and
chromosomal abnormality can occur on the same chromosome or on different
chromosomes.

[0009] Screening for other types of genetic markers are contemplated by
the methods of the invention and include but are not limited to the
detection of chemical sequence modifications. Chemical sequence
modifications include, but are not limited to, acetylation,
glycosylation, phosphorylation, and methylation. In a particular
embodiment, the methods of the invention include screening for the
presence or absence of methylation of a nucleic acid sequence, such as
de-methylation, methylation, hypomethylation and hypermethylation.

[0010] The methods of the invention are suitable for the detection of any
clinical condition or disorder. In a particular embodiment, the methods
of the invention are contemplated for use in the clinical detection of
cancer, such as breast cancer, ovarian cancer, uterine cancer, cervical
cancer, ovarian cancer, prostate cancer testicular cancer, lung cancer,
stomach cancer, brain cancer, colon cancer, kidney cancer, pancreatic
cancer, skin cancer and bladder cancer.

[0011] In a particular embodiment, the invention provides methods for
detecting cancer via the detection of a sequence mutation and loss of
heterozygosity for at least two distinct genes. The two distinct genes
may occur at different locations on the same chromosome. Alternatively,
the two distinct genes may occur on different chromosomes. In a
particular embodiment, the at least two distinct genes include FGFR3 and
p53, where a sequence mutation in FGFR3 is detected and LOH in p53 is
detected. Hypermethylation of one or more of the same or different gene
sequences can optionally be detected in combination with the gene
sequence mutation and LOH.

[0012] The methods of the invention are preferably performed in a single
assay format. In one embodiment, the screening assay is a sequencing
assay. Suitable sequencing methods include, but are not limited to,
single molecule sequencing techniques. Alternatively, the methods of the
invention can be performed using one or more methods such as real-time or
quantitative PCR, digital PCR, and/or PCR in flowing or stationary
droplets, well plates, slugs or fluid flowing segments, and the like,
microarrays for subsequent fluorescent or non-fluorescent detection,
barcode mass detection using a mass spectrometric methods, detection of
emitted radiowaves, detection of scattered light from aligned barcodes,
fluorescence detection using quantitative PCR or digital PCR methods,
Northern blot, selective hybridization, cleaved amplified polymorphic
sequence analysis, short tandem repeat analysis, the use of supports
coated with oligonucleotide probes, amplification of the nucleic acid by
RT-PCR, quantitative PCR or ligation-PCR, etc.

[0013] These and other aspects of the invention are described in further
detail in the description and claims that follow.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Methods of the invention provide a sensitive and specific test for
detecting and diagnosing different diseases or disorders, particularly
cancer. The invention recognizes that a single type of genetic
information may be insufficient for diagnosis and classification of a
disease or disorder. Rather, the assessment of a combination of different
types of genetic markers, provides a much more robust analysis tool.

[0015] Methods of the invention rely on the detection of different types
of genetic markers in order to achieve superior diagnostic accuracy. The
different types of genetic markers measured may occur on the same
chromosome, or on different chromosomes. Preferably, the detection of the
different types of genetic markers is achieved in a single assay format.

[0016] In certain aspects, both a sequence mutation and a chromosomal
abnormality is detected from a patient sample. The detection of a
sequence mutation alone may not be predictive because single biomarkers
oftentimes have a high false positive or false negative rate. In
combination with the detection of chromosomal abnormality, the desired
predictive values are achieved. The sequence mutation and the chromosomal
abnormality may occur on the same chromosome, or on different
chromosomes. Optionally, one or more types of chemical sequence
modifications (e.g., hypermethylation) may be detected in combination
with a sequence mutation and chromosomal abnormality to further improve
diagnostic accuracy.

[0017] Accordingly, methods of the invention provide for a evaluating a
patient sample for any combination of two or more characteristics in
order to form a more complete diagnostic profile for a clinical condition
or disorder.

Obtaining a Biological Sample

[0018] Methods of the invention involve obtaining a biological sample,
from a subject. Samples may include any bodily fluid such as blood, a
blood fraction, saliva, sputum, urine, semen, transvaginal fluid,
cerebrospinal fluid, or stool. Other such samples may include one or more
cells or a tissue biopsy, such as a cell or biopsy from the brain, mouth,
throat, esophagus, stomach, lymph node, stomach, intestine (large or
small), kidney, bladder, liver, pancreas, skin, muscle, bone, bone
marrow, breast, ovary, vagina, cervix, uterus, testicle or prostate.

[0020] Nucleic acids may be obtained by methods known in the art.
Generally, nucleic acids can be extracted from a biological sample by a
variety of techniques such as those described by Maniatis, et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp.
280-281, (1982), the contents of which is incorporated by reference
herein in its entirety. The isolated nucleic acid molecules may be
single-stranded, double-stranded, or double-stranded with single-stranded
regions (for example, stem- and loop-structures). The isolated nucleic
acid can be deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). In
a particular embodiment, genomic DNA is isolated from the biological
sample.

[0021] It may be necessary to first prepare an extract of the cell and
then perform further steps--i.e., differential precipitation, column
chromatography, extraction with organic solvents and the like--in order
to obtain a sufficiently pure preparation of nucleic acid. Extracts may
be prepared using standard techniques in the art, for example, by
chemical or mechanical lysis of the cell. Extracts then may be further
treated, for example, by filtration and/or centrifugation and/or with
chaotropic salts such as guanidinium isothiocyanate or urea or with
organic solvents such as phenol and/or HCCl3 to denature any
contaminating and potentially interfering proteins.

Genetic Markers

[0022] Methods of the invention involve the detection of at least two
types of genetic markers from the patient's isolated nucleic acid. The
different genetic markers preferably include a sequence mutation in one
or more genes, and partial or whole chromosomal abnormalities.

[0023] Sequence mutations contemplated by the present invention include,
point mutations such as deletions, insertions, transitions,
transversions, frameshift mutations, nonsense mutations, missense
mutations, single nucleotide polymorphisms. Methods of the invention
additionally contemplate the presence of viral DNA insertions in patient
sequences. In certain embodiments the methods of the invention are used
to identify nucleic acid sequence alterations that are causally
implicated in cancer.

[0025] LOH is a common occurrence in patients with cancer, and indicates
the absence of a functional tumor suppressor gene in the lost region.
Many people with LOH remain healthy because there still is one functional
gene left on the other chromosome of the chromosome pair. However, the
remaining copy of the tumor suppressor gene can be inactivated by a point
mutation, leaving no tumor suppressor gene to protect the body and result
in the LOH phenotype.

[0026] LOI is a result of the loss of the normal function of an imprinted
gene. Genomic imprinting is a genetic phenomenon wherein genes are
expressed in a parent-of-origin-specific manner. Imprinted alleles are
silenced such that the genes are either expressed only from the
non-imprinted allele inherited from the mother (e.g. H19 or CDKN1C), or
in other instances from the non-imprinted allele inherited from the
father (e.g. IGF-2). Genomic imprinting is an epigenetic process that
involves methylation and histone modifications in order to achieve
monoallelic gene expression without altering the genetic sequence. These
epigenetic marks are established in the germline and are maintained
throughout all somatic cells of an organism. Appropriate expression of
imprinted genes is important for normal development, with numerous
genetic diseases associated with imprinting defects including
Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman syndrome
and Prader-Willi syndrome. LOI has been implicated causally in various
cancers, including, but not limited to, breast and ovarian cancers. LOI
can be detected through the sequencing as described in S.R. Bischoff, et
al (Biol Reprod. 2009 November; 81(5): 906-920).

[0027] Screening for additional types of genetic markers may optionally be
combined with the detection of one or more sequence mutations and
chromosomal abnormalities. In certain aspects, the methods of the
invention include the detection of a sequence mutation, a chromosomal
abnormality and a chemical sequence modification. Chemical sequence
modifications include, but are not limited to, acetylation,
glycosylation, phosphorylation, and methylation.

[0028] In a particular embodiment, the methods of the invention optionally
include screening for the presence or absence of methylation of a nucleic
acid sequence, such as de-methylation, methylation, hypomethylation and
hypermethylation. DNA methylation is an important regulator of gene
transcription and a large body of evidence has demonstrated that aberrant
DNA methylation is associated with unscheduled gene silencing, and the
genes with high levels of 5-methylcytosine in their promoter region are
transcriptionally silent. Aberrant DNA methylation patterns have been
associated with a large number of human malignancies and found in two
distinct forms: hypermethylation and hypomethylation compared to normal
tissue. Hypermethylation is one of the major epigenetic modifications
that repress transcription via promoter region of tumour suppressor
genes. Hypermethylation typically occurs at CpG islands in the promoter
region and is associated with gene inactivation. Global hypomethylation
has also been shown to be causally related to the development and
progression of cancer through different mechanisms. Other chemical
modifications to the DNA that are causally related to cancer will be
known to those in the art.

Detection of Genetic Markers

[0029] Any one or combination of methods may be used for detecting the
different types of genetic markers from the patient's isolated nucleic
acid. Suitable methods include real-time or quantitative PCR, digital
PCR, PCR in flowing or stationary droplets, well plates, slugs or fluid
flowing segments, and the like, in capillary tubes, microfluidic chips,
or standard thermocycler based PCR methods known to those having ordinary
skill in the art. Additional detection methods can utilize binding to
microarrays for subsequent fluorescent or non-fluorescent detection,
barcode mass detection using a mass spectrometric methods, detection of
emitted radiowaves, detection of scattered light from aligned barcodes,
fluorescence detection using quantitative PCR or digital PCR methods.

[0030] Still other techniques include, for example, Northern blot,
selective hybridization, cleaved amplified polymorphic sequence analysis,
short tandem repeat analysis, the use of supports coated with
oligonucleotide probes, amplification of the nucleic acid by RT-PCR,
quantitative PCR or ligation-PCR, etc. These methods can include the use
of a nucleic acid probe (for example, an oligonucleotide) that can
selectively or specifically detect the target nucleic acid in the sample
to detect changes at the level of a single nucleotide polymorphism, whole
DNA-fingerprint analysis, allele specific analysis. Amplification is
accomplished according to various methods known to the person skilled in
the art, such as PCR, LCR, transcription-mediated amplification (TMA),
strand-displacement amplification (SDA), NASBA, the use of
allele-specific oligonucleotides (ASO), allele-specific amplification,
Southern blot, single-strand conformational analysis (SSCA), in-situ
hybridization (e.g., FISH), migration on a gel, heteroduplex analysis,
etc. If necessary, the quantity of nucleic acid detected can be compared
to a reference value, for example a median or mean value observed in
patients who do not have cancer, or to a value measured in parallel in a
non-cancerous sample. Thus, it is possible to demonstrate a variation in
the level of expression.

[0031] Preferably, the detection of the different types of genetic markers
is achieved in a single assay format. In a particular embodiment, the
different types of genetic markers are detected via sequencing (e.g.,
single molecule sequencing).

[0032] Sequencing may be achieved by any method known in the art. DNA
sequencing techniques include classic di-deoxy sequencing reactions
(Sanger method) using labeled terminators or primers and gel separation
in slab or capillary, sequencing by synthesis using reversibly terminated
labeled nucleotides, pyrosequencing, 454 sequencing, allele specific
hybridization to a library of labeled oligonucleotide probes, sequencing
by synthesis using allele specific hybridization to a library of labeled
clones that is followed by ligation, real time monitoring of the
incorporation of labeled nucleotides during a polymerization step, polony
sequencing, and SOLiD sequencing. Sequencing of separated molecules has
more recently been demonstrated by sequential or single extension
reactions using polymerases or ligases as well as by single or sequential
differential hybridizations with libraries of probes.

[0033] A sequencing technology that can be used in the methods of the
invention includes, for example, Helicos True Single Molecule Sequencing
(tSMS) (Harris T. D. et al. (2008) Science 320:106-109). In the tSMS
technique, a DNA sample is cleaved into strands of approximately 100 to
200 nucleotides, and a polyA sequence is added to the 3' end of each DNA
strand. Each strand is labeled by the addition of a fluorescently labeled
adenosine nucleotide. The DNA strands are then hybridized to a flow cell,
which contains millions of oligo-T capture sites that are immobilized to
the flow cell surface. The templates can be at a density of about 100
million templates/cm2. The flow cell is then loaded into an instrument,
e.g., HeliScope® sequencer, and a laser illuminates the surface of the
flow cell, revealing the position of each template. A CCD camera can map
the position of the templates on the flow cell surface. The template
fluorescent label is then cleaved and washed away. The sequencing
reaction begins by introducing a DNA polymerase and a fluorescently
labeled nucleotide. The oligo-T nucleic acid serves as a primer. The
polymerase incorporates the labeled nucleotides to the primer in a
template directed manner. The polymerase and unincorporated nucleotides
are removed. The templates that have directed incorporation of the
fluorescently labeled nucleotide are detected by imaging the flow cell
surface. After imaging, a cleavage step removes the fluorescent label,
and the process is repeated with other fluorescently labeled nucleotides
until the desired read length is achieved. Sequence information is
collected with each nucleotide addition step. Further description of tSMS
is shown for example in Lapidus et al. (U.S. Pat. No. 7,169,560), Lapidus
et al. (U.S. patent application number 2009/0191565), Quake et al. (U.S.
Pat. No. 6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S.
patent application number 2002/0164629), and Braslaysky, et al., PNAS
(USA), 100: 3960-3964 (2003), the contents of each of these references is
incorporated by reference herein in its entirety.

[0034] Another example of a sequencing technology that can be used in the
methods of the invention is 454 sequencing (Roche) (Margulies, M et al.
2005, Nature, 437, 376-380). 454 sequencing involves two steps. In the
first step, DNA is sheared into fragments of approximately 300-800 base
pairs, and the fragments are blunt ended. Oligonucleotide adaptors are
then ligated to the ends of the fragments. The adaptors serve as primers
for amplification and sequencing of the fragments. The fragments can be
attached to DNA capture beads, e.g., streptavidin-coated beads using,
e.g., Adaptor B, which contains 5'-biotin tag. The fragments attached to
the beads are PCR amplified within droplets of an oil-water emulsion. The
result is multiple copies of clonally amplified DNA fragments on each
bead. In the second step, the beads are captured in wells (pico-liter
sized). Pyrosequencing is performed on each DNA fragment in parallel.
Addition of one or more nucleotides generates a light signal that is
recorded by a CCD camera in a sequencing instrument. The signal strength
is proportional to the number of nucleotides incorporated. Pyrosequencing
makes use of pyrophosphate (PPi) which is released upon nucleotide
addition. PPi is converted to ATP by ATP sulfurylase in the presence of
adenosine 5' phosphosulfate. Luciferase uses ATP to convert luciferin to
oxyluciferin, and this reaction generates light that is detected and
analyzed.

[0035] Another example of a sequencing technology that can be used in the
methods of the invention is SOLiD technology (Applied Biosystems). In
SOLiD sequencing, genomic DNA is sheared into fragments, and adaptors are
attached to the 5' and 3' ends of the fragments to generate a fragment
library. Alternatively, internal adaptors can be introduced by ligating
adaptors to the 5' and 3' ends of the fragments, circularizing the
fragments, digesting the circularized fragment to generate an internal
adaptor, and attaching adaptors to the 5' and 3' ends of the resulting
fragments to generate a mate-paired library. Next, clonal bead
populations are prepared in microreactors containing beads, primers,
template, and PCR components. Following PCR, the templates are denatured
and beads are enriched to separate the beads with extended templates.
Templates on the selected beads are subjected to a 3' modification that
permits bonding to a glass slide. The sequence can be determined by
sequential hybridization and ligation of partially random
oligonucleotides with a central determined base (or pair of bases) that
is identified by a specific fluorophore. After a color is recorded, the
ligated oligonucleotide is cleaved and removed and the process is then
repeated.

[0036] Another example of a sequencing technology that can be used in the
methods of the invention is Ion Torrent sequencing (U.S. patent
application numbers 2009/0026082, 2009/0127589, 2010/0035252,
2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559),
2010/0300895, 2010/0301398, and 2010/0304982), the content of each of
which is incorporated by reference herein in its entirety. In Ion Torrent
sequencing, DNA is sheared into fragments of approximately 300-800 base
pairs, and the fragments are blunt ended. Oligonucleotide adaptors are
then ligated to the ends of the fragments. The adaptors serve as primers
for amplification and sequencing of the fragments. The fragments can be
attached to a surface and is attached at a resolution such that the
fragments are individually resolvable. Addition of one or more
nucleotides releases a proton (H+), which signal detected and recorded in
a sequencing instrument. The signal strength is proportional to the
number of nucleotides incorporated.

[0037] Another example of a sequencing technology that can be used in the
methods of the invention is Illumina sequencing. Illumina sequencing is
based on the amplification of DNA on a solid surface using fold-back PCR
and anchored primers. Genomic DNA is fragmented, and adapters are added
to the 5' and 3' ends of the fragments. DNA fragments that are attached
to the surface of flow cell channels are extended and bridge amplified.
The fragments become double stranded, and the double stranded molecules
are denatured. Multiple cycles of the solid-phase amplification followed
by denaturation can create several million clusters of approximately
1,000 copies of single-stranded DNA molecules of the same template in
each channel of the flow cell. Primers, DNA polymerase and four
fluorophore-labeled, reversibly terminating nucleotides are used to
perform sequential sequencing. After nucleotide incorporation, a laser is
used to excite the fluorophores, and an image is captured and the
identity of the first base is recorded. The 3' terminators and
fluorophores from each incorporated base are removed and the
incorporation, detection and identification steps are repeated.

[0038] Another example of a sequencing technology that can be used in the
methods of the invention includes the single molecule, real-time (SMRT)
technology of Pacific Biosciences. In SMRT, each of the four DNA bases is
attached to one of four different fluorescent dyes. These dyes are
phospholinked. A single DNA polymerase is immobilized with a single
molecule of template single stranded DNA at the bottom of a zero-mode
waveguide (ZMW). A ZMW is a confinement structure which enables
observation of incorporation of a single nucleotide by DNA polymerase
against the background of fluorescent nucleotides that rapidly diffuse in
an out of the ZMW (in microseconds). It takes several milliseconds to
incorporate a nucleotide into a growing strand. During this time, the
fluorescent label is excited and produces a fluorescent signal, and the
fluorescent tag is cleaved off. Detection of the corresponding
fluorescence of the dye indicates which base was incorporated. The
process is repeated.

[0039] Another example of a sequencing technology that can be used in the
methods of the invention is nanopore sequencing (Soni G V and Meller A.
(2007) Clin Chem 53: 1996-2001). A nanopore is a small hole, of the order
of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid
and application of a potential across it results in a slight electrical
current due to conduction of ions through the nanopore. The amount of
current which flows is sensitive to the size of the nanopore. As a DNA
molecule passes through a nanopore, each nucleotide on the DNA molecule
obstructs the nanopore to a different degree. Thus, the change in the
current passing through the nanopore as the DNA molecule passes through
the nanopore represents a reading of the DNA sequence.

[0040] Another example of a sequencing technology that can be used in the
methods of the invention involves using a chemical-sensitive field effect
transistor (chemFET) array to sequence DNA (for example, as described in
US Patent Application Publication No. 20090026082). In one example of the
technique, DNA molecules can be placed into reaction chambers, and the
template molecules can be hybridized to a sequencing primer bound to a
polymerase. Incorporation of one or more triphosphates into a new nucleic
acid strand at the 3' end of the sequencing primer can be detected by a
change in current by a chemFET. An array can have multiple chemFET
sensors. In another example, single nucleic acids can be attached to
beads, and the nucleic acids can be amplified on the bead, and the
individual beads can be transferred to individual reaction chambers on a
chemFET array, with each chamber having a chemFET sensor, and the nucleic
acids can be sequenced.

[0041] Another example of a sequencing technique that can be used in the
methods of the invention involves using an electron microscope
(Moudrianakis E. N. and Beer M. Proc Natl Acad Sci USA. 1965 March;
53:564-71). In one example of the technique, individual DNA molecules are
labeled using metallic labels that are distinguishable using an electron
microscope. These molecules are then stretched on a flat surface and
imaged using an electron microscope to measure sequences.

[0042] Chemical modifications to a nucleic acid sequence can be detected
known methods in the art. In other certain aspects, DNA methylation is
detected. Methods for DNA methylation analysis can be divided roughly
into two types: global and gene-specific methylation analysis. For global
methylation analysis, there are methods which measure the overall level
of methyl cytosines in genome such as chromatographic methods and methyl
accepting capacity assay.

[0043] For gene-specific methylation analysis, a large number of
techniques have been developed. Most early studies used methylation
sensitive restriction enzymes to digest DNA followed by Southern
detection or PCR amplification. Recently, bisulfite reaction based
methods have become very popular such as methylation specific PCR (MSP),
bisulfite genomic sequencing PCR. Bisulfite Modification (Conversion)
uses sodium bisulfite to convert unmethylated cytosines to uracils and
subsequently detects methylated cytosines using methylation specific PCR
(MSP) technique or bisulfite genomic sequencing after PCR amplification
with or without cloning. Bisulfite genomic sequencing allows precise
analysis of methylation in a certain region by converting all
nonmethylated cytosines into thymines, while methylated cytosines remain
unchanged. This method requires small amount of genomic DNA and therefore
seems to be very useful for the analysis of clinical samples, where the
material amount is limited. A protocol has been developed for handling
small numbers of cells and little/limited DNA. The protocol is based on a
strategy using agarose embedded DNA. This physical trapping helps to
avoid DNA loss during the various incubation steps while maintaining a
good bisulphite conversion rate.

[0044] Additionally, in order to identify unknown methylation hot-spots or
methylated CpG islands in the genome, several of genome-wide screen
methods have been invented such as Restriction Landmark Genomic Scanning
for Methylation (RLGS-M), and CpG island microarray.

[0047] References and citations to other documents, such as patents,
patent applications, patent publications, journals, books, web contents,
have been made throughout this disclosure. All documents are hereby
incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

[0048] The invention may be embodies in other specific forms without
departing from the spirit or essential characteristics thereof. The
foregoing embodiments are therefore to be considered in all respects
illustrative rather than limiting on the invention described herein.
Scope of the invention is thus indicated by the appended claims rather
than by the foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore intended to
be embraced from within.